Project Summary/Abstract Nerve injury-induced neuropathic pain is a major health problem with limited treatment. Insufficient understanding of this disorder at the level of neural circuits impedes the development of effective therapies. The activation of the immune system and neuronal alterations in the somatosensory pathway are known to be critical for the genesis of neuropathic pain. However, how immune cells interact with neurons after peripheral nerve injury and contribute to persistent and intense pain sensation remains largely elusive. Our recent studies have shown that monocytes and microglia synergistically promote the development of neuropathic pain symptoms. We have also developed novel approaches for imaging and manipulating neurons and nonneuronal cells (e.g. monocytes, microglia and astrocytes) in the pain pathway, from peripheral dorsal root ganglia (DRG), the primary somatosensory cortex (S1) to anterior cingulate cortex (ACC) in the awake, behaving mice. In our preliminary studies, we have found that following peripheral nerve injury, DRG sensory neuron activities undergo rapid and transient increases whereas pyramidal neuronal activities in the S1 and ACC rise progressively and remain elevated under chronic pain states. Moreover, selective depletion of microglial cytokine TNF-alpha production attenuates pain hypersensitivity after nerve injury. Based on these findings, we hypothesize that the interactions among peripheral monocytes, brain microglia and astrocytes play critical roles in the emergence of hyperactive neurons in the cortex during the transition from acute to chronic pain. In this application, we will test this hypothesis by combining in vivo two-photon imaging of synapse structure and neuronal activity, genetic manipulation of molecularly defined cell types in cortical circuitry, as well as behavioral testing for sensory and affective pain symptoms. Specifically, in Aim 1, we will characterize the sequential changes of neuronal activities in the pain circuits after peripheral nerve injury. This will test the hypothesis that dynamic changes in pain circuits occur progressively from the DRG to the cortex and that persistent neuronal hyperactivity emerging in S1 and ACC is critical for the induction of chronic neuropathic pain. In Aim 2, we will combine in vivo calcium imaging with in vivo cell depletion and bone marrow chimeric strategies to investigate the synergistic roles of monocytes and microglia in the emergence of persistent cortical hyperactivity. In Aim 3, we will test the hypothesis that monocytes/microglia promote nerve injury- induced neuronal hyperactivity via proinflammatory cytokines and astrocytes in the cortex. Together, our proposed research will significantly expand the knowledge on the contribution of neuroimmune interactions to the development of neuropathic pain. They will also help identify immune cells as novel targets for the development of effective pain therapies.